Neoplastic transformation of higher plant cells is conferred by genes inserted during infection of plants by Agrobacterium tumefaciens. These genes (estimated to be 6-10 genes) are situated on a 120 megadalton plasmid that is harbored in this bacterium. They are stably maintained indefinitely in tumor cells in axenic culture. The precise location of these plasmid sequences in the tumor cells is unkown and experiments are proposed to determine whether or not they are associated with organelle or nuclear DNA. These experiments will involve DNA.DNA solution hybridization and Southern blot hybridization and analysis with labelled plasmid DNA and highly purified organelle and nuclear DNA. The tumor cell plasmid sequences will also be located on a physical map of the large plasmid using the transposition element Tn7 to obtain mutants with insertionally inactivated oncogenicity and other phenotypic characters, followed by analysis of overlapping plasmid segments generated by restriction endonucleases using reciprocal digestions with two or more different restriction enzymes. Several phenotypic characters known to be specified by the plasmid will be used as markers in the map. These include oncogenicity, transfer function, octopine or nopaline utilization, octopine or nopaline biosynthesis, phage and bacteriocin sensitivities and synthesis of periplasmic proteins Per-1 and Per-2. Special emphasis will be given to determine whether or not the regulation or biosynthesis of the plant growth hormone, indoleacetic acid, is specified by the plasmid sequences located in tumor cells. This will be accomplished by genetic experiments using Tn7 and biochemical analysis of insertional mutants. Also special efforts will be made to determine the nature of the newly discovered anti-tumor agent induced by RNA virus infection. This will involve isolation, purification and characterization of the compound. The mechanism of action of this compound fication and will be studied through its interaction with plasmid DNA transfer, plasmid sequences and plasmid gene products as some of the possibilities. All of the above studies will provide us with a substantial data base for understanding the mechanism of tumorigenesis.